Phytases (myo-inositol hexakisphosphate phosphohydrolase) [EC 3.1.3.26 and EC 3.1.3.8] are phosphatases that initiate the sequential liberation of orthophosphate groups from phytate (InsP6, myo-inositol 1,2,3,4,5,6-hexakisphosphate), providing phosphate, inositol phosphates and inositol required for a range of cellular activities (Brinch-Pedersen et al., 2002). A number of enzymes with phytase activity are known from plants, animals and microorganisms (Dvorakova, 1998).
Phytases are of particular importance during seed germination where they mobilize phosphate from phytate, the major reserve of phosphorus (P) in plant seeds accounting for ˜70% of the total P (Lott, 1984). Different plant species have developed various strategies for phytase mediated degradation of phytate during germination. Among cereals, barley (Hordeum vulgare L.), wheat (Triticum aestivum and durum L.) and rye (Secale cereale L.) synthesize and accumulate phytase during grain development and the mature seed has a significant level of preformed phytase activity. Levels of phytase activity of 582, 1193 and 5130 U kg−1 have been detected in mature grain of barley, wheat and rye respectively (Eeckhout and de Paepe, 1994). Preformed phytase catalyses the first wave of phytate hydrolysis during early germination. Other cereals possess little (maize (Zea mays L.) ˜41 U kg−1) or close to no (rice (Oryza sativa L.)) preformed phytase activity in the mature seed and depend entirely on de novo synthesis during germination (Eeckhout and de Paepe, 1994).
The spatial and temporal regulation of phytase biosynthesis in plant seeds has profound effects on phosphate bioavailability when dry grains are used as food and feed. Monogastric animals such as pigs, poultry and humans have little or no phytase activity in their digestive tracts and thus depend on either a phosphate supplement or on the presence of the enzyme phytase in their diet, in order to meet their nutritional phosphate requirements. In most cases the amount of preformed phytase activity in mature cereal grain is not sufficient to ensure sufficient phytate degradation when included in animal feed. As a consequence, most of the cereal grain phytate consumed by an animal is excreted, thereby adding to the phosphate load on the environment which can be massive in areas with intense livestock production. One current solution to this problem has been to supplement animal feed, on a large scale, with inorganic phosphate, in order to meet an animal's need for phosphate. However, this solution can only continue in the short term since phosphate is a non-renewable resource, which will be depleted within a few decades. An alternative solution relies on the addition of phytase enzyme, in particular microbial-derived phytase, to feed intended for intense pig and poultry production. It has become common practise to include the enzyme phytase in pre-mixes for addition to animal fodder, and animal fodder, which is an additional cost factor. Thus there exists a need for alternative cheaper methods for enhancing the bioavailability of phosphate in cereals used for animal feed.
A DNA sequence comprising a coding sequence for wheat phytase has been deposited in GenBank (AX298209). Patent application (WO2001/083763A2) describes said wheat phytase as a 66 kDa PAPhy with the same temperature and pH optima as PHYI (Rasmussen et al., 2004), and describes the production of transgenic wheat plants comprising said coding sequence.